Melanoma

New research is revealing more options to target melanoma cancer cells to control tumor growth and advancement. Photo: The Horse Staff

I was like a kid in a candy shop, having just shopped for and found my horse of a lifetime. My new gray 8-year-old Arabian was perfect in almost every respect, but he came to me with a large, ulcerated cauliflowerlike mass to the left of his tailhead, oozing black tarry material.

This was back in the mid-­­1980s, when I was a recent veterinary school graduate, and we didn’t have many options for dealing with equine melanoma other than to take out the scalpel and get to work. After I’d excised the cutaneous (affecting the skin) lesion, all seemed well. But two years later, when loading him on the trailer I noticed an abnormal out-pouching alongside his anus as he defecated. On rectal exam I felt an orange-sized mass directly internal to where the original lesion had been. Three weeks later, on another rectal exam, I discovered that it had grown to grapefruit size. My veterinarian partner and I laid my horse down on the surgical table under anesthesia and cut it out, finding it extended deep inside along his small colon and rectum.

At that time a newly proposed melanoma treatment had surfaced: using oral cimetidine to activate one arm of the immune system—T-cells—to keep the cancer in check. Not then available as a generic drug, it was expensive. And, of course, my finicky Arabian managed to pick out all the ground-up tablets from his food unless I mixed them thoroughly with applesauce. Researchers have since studied cimetidine, only to find inconsistent results for melanoma management. I have no way of knowing if the surgical procedure did the trick or if the cimetidine helped, but my horse lived until almost 29, dying of other causes.

Equine medicine has come a long way since then, bringing an entirely new arsenal of approaches to issues like equine melanoma. The literature reports that 80% of gray horses over the age of 15 are likely to end up with melanoma. Small nodular lesions that are more of a cosmetic problem develop in some, while aggressive tumors that are potentially invasive to internal organs develop in others. Currently, there are two melanoma “vaccines” under investigation and being considered for USDA approval, which we will discuss here. These vaccines are not meant as protection against developing melanoma lesions but, rather, as treatments for already-existing cancer.

IFx-VETDirect Immunotherapy

Cells that develop cancer are aberrations of normal cells in an animal’s body and, therefore, are not recognized as foreign.  This enables cancer cells to escape immune system detection and continue to divide and spread, advancing and taking over normal tissue. When looking for that something special that can break these tumor cells’ tolerance, the objective is to find a substance that causes the tumor to express antigens (foreign protein that evokes an immune response), so the animal’s native immune system recognizes the cancer as something it must eliminate. Researchers at Morphogenesis Inc., in Tampa, Florida, have identified a bacterial gene they say “primes and educates the immune system to attack tumor cells throughout the body.” In so doing, it causes no harm or damage to healthy cells or tissues.

Pat Lawman has her PhD in immunology and molecular biology and is CEO of Morphogenesis Inc. She explains that the insertion of a single bacterial gene (plasmid DNA) into tumor cells produces highly immunogenic cell proteins on their surface. “When the immune system sees this antigen, it cannot ignore it,” she says. “This then becomes the crux of the immune response: All tumor antigens are presented to the immune system in an appropriate way.”

A veterinarian inserts the specialized bacterial gene directly into the tumor in the living horse. Morphogenesis Inc. derives the product, known as IFx-VETDirect multi-indication cancer vaccine, from the bacterium Streptococcus pyogenes. Once tumor cells take up this plasmid DNA, they begin to express an antigenic protein (Emm55) that the horse’s immune system can recognize and then target.

The company’s researchers discovered that injecting the plasmid DNA directly into the melanoma results in significant tumor remission. They selected three lesions on each of 26 study horses, measuring and evaluating all lesions over 35 weeks. Four of the horses served as controls and were injected with buffer only.

The protocol for direct treatment of lesions calls for administering four injections bi-weekly and then twice more monthly. In the study researchers administered the vaccine directly into the tumor using a disposable CO2-powered, needleless injector. They injected a single shot of DNA plasmid material under pressure to get the DNA through cell membranes, where it is taken up within the interstitial spaces (between cells). Once the antigen is present, says Lawman, T-cells kill the tumor locally while also eliciting a systemic response to kill cancer in noninjected tumors elsewhere on the body.

Results were encouraging: 77% of horses given the intratumoral vaccine experienced tumor regression over 35 weeks. Researchers saw no adverse effects on blood chemistries or metabolic panels, says Lawman. They noted either control of the tumor(s) or at least 30% decrease in lesion size in all 22 vaccinated horses. These were significant results, she says, especially when compared to tumor progression of the study’s four control horses.

United States Department of Agriculture approval is still in process, so this product is not yet available commercially in the mainstream equine health care market. While awaiting USDA approval, it is currently possible for veterinarians to use this “vaccine” model in an autologous procedure (produced directly from the tissue donor) that involves harvesting melanoma tissue from a horse with a cancerous lesion.

In this version, known as a whole-cell vaccine, “the veterinarian sends a sample of a patient’s tumor to our lab where we digest the tumor to obtain a cell suspension,” said Lawman. “We then insert the gene into the tumor cells via electroporation (applying an electrical field to cells to increase the permeability of the cell membrane). Once the gene is expressed, we irradiate the cells and cryopreserve them. The doses (five to eight, generally) are then sent back to the veterinarian for intradermal (into the skin) injection.”

Bryan Waldridge, DVM, MS, Dipl. ABVP, ACVIM, of Park Equine Hospital at Woodford, in Versailles, Kentucky has experience using this autologous vaccine in two horses. One of his patients is the accomplished Thoroughbred stallion Alphabet Soup, winner of the 1996 Breeders’ Cup. Waldridge reports that treatment has achieved an 80% reduction in the size of the stallion’s melanoma masses in the perineal area (around the anus). The protocol calls for four weekly injections and then another four monthly injections. The company recommends injecting the vaccine intradermally near lymph nodes, not necessarily directly into the tumors. Waldridge injected Alphabet Soup in the neck for each treatment. Despite the distance between the injection site and the back end of the horse, the tumors shrunk significantly. The other case Waldridge treated is a broodmare, also with perineal melanoma masses. He says her tumors shrunk some but then became static without as much resolution as seen in the stallion.

Oncept

Currently, there is an available USDA-approved plasmid DNA vaccine that small animal practitioners are using to treat oral melanoma in dogs; the product is called Oncept (Merial-Boehringer-Ingelheim Inc.). It does not cure a dog’s melanoma of the oral cavity, but it has proven to extend life expectancy from this aggressive canine cancer for an additional 18 to 24 months. Studies have been ongoing to evaluate its application in horses.

The DNA within the Oncept vaccine stimulates production of human tyrosinase in dogs. Tyrosinase is a specific protein that controls the production of melanin pigment in many species. Hoyt Cheramie, DVM, MS, Dipl. ACVS, senior equine professional service veterinarian for Boehringer-Ingelheim Inc. remarks that the immune system looks at this protein all the time but, because it is “self,” the dog’s immune system doesn’t act to eliminate its normal amounts. However, through vaccination, the human tyrosinase protein the dog produces acts as an antigen (“not-self”). This makes it apparent to the immune system, which recognizes it as foreign and targets it for removal along with cancerous melanoma cells, which produce significant amounts of tyrosinase. Researchers identified that the immune response to human tyrosinase is similar in both dogs and horses, thereby making this a viable product for use in equine melanoma treatment.

Cheramie describes two independent studies by veterinary oncologist Jeffrey Phillips, DVM, MSpVM, PhD, Dipl. ACVIM: In the first he used Oncept in normal horses without melanoma lesions and examined their responses. These horses mounted a measurable immune response to the vaccine, while maintaining normal function of their own tyrosinase. The second study involved 15 horses with melanoma lesions; these individuals received transdermal (through the skin) Oncept injections into pectoral muscles. Cheramie says follow-up on these horses is now into its fourth year. Morris Animal Foundation funded the initial three-year study; follow-up studies are also in the hands of Jim Blackford, DVM, MS, Dipl. ACVS at the University of Tennessee’s College of Veterinary Medicine. Blackford has tried the Oncept product on approximately 50 privately owned horses not involved in the original study.

He followed the canine protocol for the horses: Using a needleless injector device, he administered four injections every two weeks transdermally in the pectoral muscle, followed by a booster every six months. Twice yearly “boosters” are likely necessary for the life of the horse, Blackford says. The equine dose is the same as that used for dogs; he comments that researchers found that a higher dose did not necessarily improve the outcome. Blackford explains that the vaccine’s plasmid DNA replicates well in solid muscle, and transdermal administration enables it to penetrate 1½ to 2 inches into the pectoral muscle.

Most study horses–80% (13/15)–experienced a positive response. Twelve had an at least 30% reduction in tumor size. One horse did not experience tumor size reduction; however, treatment stopped rapid growth of an aggressive tumor. There were no reported significant adverse effects, just some white spots in the area of resolving melanomas. In nonstudy horses Blackford has seen occasional 2-3-millimeter swellings at the injection site, which resolve within 48 to 72 hours.

Blackford says his experience with the nonstudy horses, ranging in ages from 9 to 30, has been very favorable. All but one of the horses is gray; the other is bay. For horses with long-standing tumors treated with Oncept, he reports it’s not uncommon to see initial tumor enlargement before tumors either return to their original size and stop growing, or shrink somewhat.

“Two horses had cutaneous tumor lesions excised prior to vaccination and became tumor-free,” he says. “One was a gray with girth lesions; the other the bay with a solitary flank lesion. I suggest seeing how much change in lesion size we get with the vaccine when people choosing that modality start out talking about excision.”

Cutting out melanoma tumors could incite them to spread. He stresses that treatment must be individualized for each horse because some will have tumors recur before the six-month booster, so it might need to be given sooner.

More importantly, Blackford notes, “There is excellent success when using this Oncept vaccine early on when new melanoma lesions first appear. The full protocol of Oncept given every two weeks for four injections and then every six months has resulted in complete resolution of early tumors, with the horses remaining tumor-free.”

Also noteworthy is that following the initial four vaccine injections, seven horses in the nonstudy group experienced cutaneous rupture of the lesions. Historically, if these ruptured lesions weren’t treated in nonvaccinated horses, they would drain indefinitely. But with Oncept treatment, Blackford says they heal spontaneously without tumor recurrence. This is evidence that the antibodies the body produces in response to the vaccine are attacking the tumor, he says. One caveat is that we can’t always determine the presence of internal lesions, which could rupture internally with significant adverse consequences. He advises veterinarians to perform a full rectal exam of the pelvic canal in horses with cutaneous perineal lesions.

“If within an owner’s financial means, the best approach is to start the vaccine immediately when new cutaneous melanoma lesions are first identified,” he says. “The vaccine targets lesions on the whole horse.”

To date, this is still an experimental vaccine, not yet labeled for horses and still awaiting USDA approval for use in horses. Cheramie notes, “Study data has been submitted to the Center for Veterinary Biologics at the USDA. Discussions are underway, and decisions are pending.”

Availability of the canine vaccine is limited to board-certified small animal internal medicine and oncology specialists. Availability to equine veterinarians will be determined later if the vaccine receives approval.

Betulinic Acid

You might have heard about people in ancient civilizations chewing on birch bark (Betula sp) for its medicinal anti-inflammatory properties. Birch bark and leaves are rich in salicylic acid, a component of aspirin. And some non-steroidal anti-inflammatory medications possess anti-neoplastic properties (neoplasms being abnormal growths). With this in mind, researchers at the Martin Luther University at Halle, Germany, examined the chemotherapeutic use of betulinic acid and its derivatives on equine melanoma tissue in the lab. Once they saw melanoma-killing activity, they encapsulated the material and used it on two gray horses with melanoma lesions. They injected the formulation weekly for 19 weeks directly into each tumor.

The drug reaches the cells or attaches to them within the initial 24 hours after treatment. Cancer cell death occurs at about 48 hours after application. The researchers report no adverse effects on noncancerous cells. As scientists complete more studies on live horses, time will tell if betulinic acid will serve as another useful melanoma treatment.

Take-Home Message

While a large percentage of gray horses over the age of 15 develop localized cutaneous melanoma lesions, many tend to be cosmetic in nature. However, invasive malignant melanoma does occur. Metastases, or secondary growth at distances from the primary tumor, to internal structures such as salivary glands, the gastrointestinal tract, and the guttural pouch might cause weight loss, colic, neurologic conditions, or difficulty defecating. New research is revealing more options to target melanoma cancer cells to control tumor growth and advancement. While scientists are still in the investigative process, our sources say vaccine technology that relies on cloning DNA genes into molecular vectors shows promise for managing equine melanoma.